Soluble Toll-Like Receptor 4 Impairs the Interaction of Shiga Toxin 2a with Human Serum Amyloid P Component.

Maurizio Brigotti,Xiaohua He,Valentina Arfilli,Gaia Scavia,Stefano Morabito,Francesca Ricci,Pier Tazzari,Domenica Carnicelli,Gianluigi Ardissino

doi:10.3390/toxins10090379

Abstract

Shiga toxin 2a (Stx2a) is the main virulence factor produced by pathogenic Escherichia coli strains (Stx-producing E. coli, STEC) responsible for hemorrhagic colitis and the life-threatening sequela hemolytic uremic syndrome in children. The toxin released in the intestine by STEC targets the globotriaosylceramide receptor (Gb3Cer) present on the endothelial cells of the brain and the kidney after a transient blood phase during which Stx2a interacts with blood components, such as neutrophils, which, conversely, recognize Stx through Toll-like receptor 4 (TLR4). Among non-cellular blood constituents, human amyloid P component (HuSAP) is considered a negative modulating factor that specifically binds Stx2a and impairs its toxic action. Here, we show that the soluble extracellular domain of TLR4 inhibits the binding of Stx2a to neutrophils, assessed by indirect flow cytometric analysis. Moreover, by using human sensitive Gb3Cer-expressing cells (Raji cells) we found that the complex Stx2a/soluble TLR4 escaped from capture by HuSAP allowing the toxin to target and damage human cells, as assayed by measuring translation inhibition, the typical Stx-induced functional impairment. Thus, soluble TLR4 stood out as a positive modulating factor for Stx2a. In the paper, these findings have been discussed in the context of the pathogenesis of hemolytic uremic syndrome.

Highlights

Shiga toxins (Stx) are powerful exotoxins involved in the pathogenesis of severe illnesses in children, such as hemorrhagic colitis and hemolytic uremic syndrome (HUS), related to infections by pathogenic Escherichia coli strains (Stx-producing E. coli, STEC) [1,2,3]
Since neutrophils and other innate immunity cells bind Stx through Toll-like receptor 4 (TLR4) and become activated after toxin stimulation, we have studied the effect of 70 kDa sTLR4 on the biological activity of these toxins and on the Shiga toxin 2a (Stx2a)/human serum amyloid P component (HuSAP) interactions
Human neutrophils from healthy donors were treated with 60 nM Stx1a and Stx2a to reach full saturation of receptors [31]

Summary

Introduction

Shiga toxins (Stx) are powerful exotoxins involved in the pathogenesis of severe illnesses in children, such as hemorrhagic colitis and hemolytic uremic syndrome (HUS), related to infections by pathogenic Escherichia coli strains (Stx-producing E. coli, STEC) [1,2,3]. Stx bind to several circulating cells (precocious toxaemia) [4,5] before targeting renal and cerebral endothelia which express the high-affinity receptor of the toxin, globotriaosylceramide (Gb3Cer) [6,7]. The latter event is the crucial point in the pathogenesis of HUS and occurs during advanced toxemia. As a consequence of the resulting endothelial injury/dysfunction, the formation of microthrombi occurs in renal glomeruli, causing the HUS triad, i.e. acute renal failure, mechanical damage of erythrocytes, and thrombocytopenia [1,3]

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